The chemical synthesis of oligonucleotides of DNA fragments is performed efficiently by the phosphoramidite chemistry, and the coupling reaction gives excellent yield on various solid supports (Oligodeoxy nucleotide synthesis, Phosphoramidite Approach, Serge L. Beaucage in Protocols For OLigonucleotides and Analogs, Synthesis and Properties, Editor, Sudhir Agarwal, Humana Press, 1993). Similarly excellent protocols have been developed for the synthesis of RNA, and various biologically active tRNA molecules (Oligoribonucleotide synthesis, The Silyl Phosphoramidite Method, Masad J. Damha and Kevin K. Ogilvie in Protocols For Oligonucleotides and Analogs, Synthesis and Properties, Editor, Sudhir Agarwal, Humana Press, 1993). A large number of such biologically functional DNA and RNA molecules carry base labile and modified nucleosides which cannot sustain prolonged basic conditions, generally required during deprotection. Thus dihydrouridine present in tRNA requires very mild deprotection conditions, otherwise it is completely decomposed and the quality of synthetic tRNA will be compromised. (C. Chaix, D. Molko and R. Teoule, Tetrahedron Letters, 30, 1, 711-74, 1989). In order to develop protecting groups which are milder in nature have been developed in recent past. Thus 2-(acetoxy-methyl)benzoyl (AMB) group has been reported which uses potassium carbonate as mild deprotecting group for their removal (W. H. A. Kuijpers, J. Huskens and C. A. A. Van Boeckel, Tetrahedron Lett., 31, 6729-6732, 1990) & W. H. A. Kuijpers, E. Kuyl-Yeheskiely, J. H. Van Boom and C. A. A. Van Boeckel, Nucl. Acids Res., 21, 3493-3500, 1993). The AMB group seems attractive, however there are many practical problems in their use. FMOC group was reported for the protection of amino function of 2′-deoxycytidine, 2′-deoxy adenosine and 2′-deoxy guanosine and for the corresponding ribonucleosides (H. Heikkila and J. Chattopadhyaya, Acta Chem. Scand. B 37, No. 3, 263-265, 1983). FMOC as n-protecting group as pointed out by these authors for their capability to get cleaved under very mild alkaline deprotection condition, or by bases capable to carry out selective deprotection via B-elimination of FMOC group (scheme 1). It is therefore not surprising that other attempts to synthesis of N-FMOC protected nucleoside and phosphoramidites have been carried out. The publication (R. K. Gaur, V. Bobde, M. Atreyi and K. C. Gupta, Indian Journal of Chemistry, 29B, 108-112, 1990), reports preparation of 5′-DMT-n-FMOC-dA (structure 1) and 5′-DMT-n-FMOC-dC (structure 2). However these authors could not synthesize 5′-DMT-N-FMOC-dG (structure 5). Further only p-methoxy phosphoramidites of 5% DMT-n-FMOC-dC (structure 3) and 5′-DMT-n-FMOC d A (structure 4) were synthesized by these authors. The p-methoxy phosphoramidites have only limited application in oligonucleotide synthesis. Further, no N-FMOC protected solid supports were reported.

The synthesis of N-FMOC-5′-DMT-dG (structure 5) had eluded so far till our present invention.

Similarly no solid supports or the succinates of the N-FMOC-5′-DMT-deoxy bases (dA and dC) revealed by these other authors. Hence there is no information as to the applicability of DMT-N-6-FMOC-dA-3′-succinyl-support, which would validate the concept that the solid support containing N-6-Fmoc-dA solid supports are expected to minimize or dramatically reduce formation of N-1, i.e. depurination of 3′-dA base products during oligo synthesis and hence about the quality of the synthesized 2′-deoxy oligonucleotides. The FMOC protected nucleoside products improve the quality of the terminal 3′-dA containing oligonucleotides.
There seem to be no further attempts to make 5′-DMT-N-FMOC dG or the ribonucleoside containing N-Fmoc protected RNA synthesis synthons and the corresponding cyanoethyl phosphoramidites. The cyanoethyl phosphoramidite chemistry in DNA and RNA are currently used in present state of the art in this technology.
It was demonstrated by the authors (J. Heikkila and J. Chattopadhyaya, Acta Chem. Scand. B 37, No. 3, 263-265, 1983) that deprotection of FMOC protecting group can be carried out under various very mild basic reaction conditions. It is possible to utilize either aq ammonia condition deprotection, which results in nucleophilic displacement of FMOC protecting group, or by a Non-nucleophilic base such as triethylamine, which causes B-elimination of FMOC-active hydrogen group (scheme 1). These authors however similarly did not pursue the n-FMOC protecting group for further exploration and carried out investigation of other protecting groups such as ortho-nitro phenyl sulfenyl protected nucleosides (Structure 6).
The FMOC protecting group is very well established in peptide synthesis and one of the preferred reagents for amino group protection of alpha-amino group of amino acids for step wise peptide synthesis (Carpino, L. A., and Han, G. Y., J. Amer. Chem. Soc., 92, 5748, 1970). But Heikkila and Chattopadyaya (J. Heikkila and J. Chattopadhyaya, Acta Chem. Scand. B 37, No. 3, 263-265, 1983), who initially synthesized the FMOC deoxy and ribo nucleosides switched to (J. Heikkila, N. Balgobin and J. Chattopadhyaya, Acta Chem. Scand B 37, 857-864, 1983) to another N-protecting group, 2-nitrophenyl sulfenyl (Nps) for the protection of amino function of cytidine, adenosine, guanosine and the corresponding 2′-deoxy ribo nucleosides (structure 6).


It is well known that cyanoethyl protecting group for internucleotide phosphate is eliminated by B-elimination mechanism leading to acrylonitrile and phosphodiester oligonucleotides. (scheme 2).

It is therefore possible to modulate the FMOC protecting group removal conditions from oligonucleotides, and the FMOC as base protecting group can be removed by the process of B-elimination, just like the B-elimination process to remove cyanoethyl group.
This process therefore offers very attractive potential to use ammonia free oligo synthesis. This process, furthermore, has potential to offer deoxyoligonucleotides for complete deprotection of oligos on solid supports. This technology or process has the potential to offer ribonucleotides such as those required for chip based technology as well high purity oligonucleotides for microRNA, Si RNA, RNA chips
This group, in conjunction with cyanoethyl phosphate protecting group, therefore offers opportunity to remove both FMOC and cyanoethyl groups from the synthesized deoxy and ribo oligonucleotides on the support cleanly, preferably with non aq bases, and on support for many diagnostics application.
Our present invention is revealed by structures such as 7-10 below.
The present inventors recently proposed to utilize N-FMOC protected purine and pyrimidine bases for the Sythesis of protected Deoxy nucleosides, ribonucleosides, phosphoromidites and their use in the synthesis of oligonucleotides. Although the FMOC protecting group presents tremendous advantage, practical large scale synthesis is inconvenient and difficult to achieve in order to produce large quantities of therapeutic grade RNA or RNA chimeras having sensitive groups such as 2′-fluoro group. It is also significantly labile so that this group has a tendency to fall off and lead to byproducts or impurities. We therefore turned our attention to N-2-acetyl as guanine base protecting group. This group is significantly more stable than FMOC protecting group. Yet is quite labile as compared to isobutyryl group at N-2 position.
Our results of RNA synthesis show that quality of RNA's suffer heavily if in the automated RNA synthesis on DNA/RNA synthesizer, DMT-N-2-isobutyryl guanosine-2′-TBDMS-3′-Cyanoethyl phosphoramidite is utilized. The currently utilized, most popular and industry standard N-2-amino protecting group for guanine bases is isobutyryl. On the other hand, if DMT-N-2-acetyl guanosine-2′-TBDMS-3′-Cyanoethyl phosphoramidite (structure 6) is used, high quality of RNA's are obtained. This has been substantiated by oligonucleotide Mass spectral data presented in the present invention.
The deprotection kinetics of N-2 acetyl guanine in a nucleoside or oligionucleotide is significantly faster as compared to standard N-2 isobutyryl group on guanine residue, so that the quality of oligonucleotides especially RNA and modified RNA increases dramatically.



The utility of N-FMOC protected nucleoside was discovered by the present inventors and it was shown to possess additional significance and importance. When oligo ribonucleotide chimeras comprise of mixed bases composed of 2′-fluoro and 2′-ribo bases they present a challenge in obtaining pure chimera oligonucleotides. It has been well documented by several recent reports that oligonucleotide chimeras having 2′-fluoro-2;′-deoxy bases along with natural ribo bases present difficulty in obtaining pure oligos. It has been shown that with strongly basic conditions, there is significant loss of fluorine as loss of Hydrogen Fluoride (“HF”) is seen as M-20 peak in Mass spectral analysis. Besides it has also been shown that uracil and cytosine are eliminated to a significant extent, when oligo chimeras containing 2′-fluoro-2′-deoxy uridine and 2′-fluoro-2′-deoxy cytidine are part of chimeras (see scheme 3).

The studies as shown in scheme 3 were carried out independently by two groups recently. Ken Hill, Agilent Technologies, Boulder, Colarado; Identification of Process Related Impurities—Understanding Oligonucleotide Production, TIDES 2007, Las Vegas. Nev. The author showed depyrimidation of chimera oligonucleotides carrying 2′-fluoro-2′-deoxy pyrimidine in RNA's; and,
Nanda D. Sinha, Avecia Biotechnologies Inc., Massachusetts—Depyrimidation, as well as loss of HF and chain cleavage in chimeras having 2′-fluoro-2′-deoxy pyrimidines in RNA sequences. Eurotides, 2005, Munich, Germany.
Our data shows that by utilizing n-acetyl-guanine protected ribonucleoside phosphoramidites, used in oligo with 2′-fluoro substitution, high quality full length RNA were obtained.
It is therefore imperative to utilize 2′-fluoro-2′-deoxy nucleosides and corresponding phosphoramidites with guanine protecting groups having N-2 acetyl guanine for mild and shorter base deprotection protocol. Although there is no B-elimination pathway for N-2-acetyl guanosine protecting group, still high purity and integrity oligo chimeras is obtained and RNA high quality for therapeutic and diagnostic applications, such as for applications in SiRNA synthesis.
Besides the 2′-fluoro nucleosides, 2′-O-alkyl nucleoside phosphoramidites are extensively used in the design of biologically active oligonuceotides for therapeutic and diagnostic applications as fully alkylated or as chimeras. Amongst the 2′-O-alkyl nucleosides and phosphoramidites the most common are 2′-O-Methyl oligonucleotides which have shown enormous promise in drug design and specific diagnostics applications. Thus 2′-Omethyl oligoribonucleotides-RNA complexes have higher Tm than corresponding oligo-deoxy ribonucleoside—RNA duplexes, Iribarren, A. M., Sproat, B. S., Neuner, P., Sulston, I., Ryder, U., and Lamond, A. I., Proc. Natl. Acad. Sci. USA 87, 7747-7751, 1990. Various 2′-OMethyl-N-FMOC protected nucleosides and phosphoramidites offer great advantage to produce high quality of DNA-RNA oligonucleotides and chimera for biological applications.
Defined sequence RNA synthesis is now well established and currently in use for synthesis and development of vast variety of therapeutic grade RNA aptamers, tRNA's, Si RNA and biologically active RNA molecules. This approach utilizes a ribonucleoside with suitable N-protecting group, 5′-Protecting group, generally, and most popular being dimethoxytriphenyl, commonly called DMT group, 2′-protecting group, out of which most popular is t-Butyldimethylsilyl ether and a 3′-phosphoramidite, wherein the most popular still is cyanoethyl diisopropyl (component 1). This component is then coupled with a nucleoside with a suitable N-protecting group, 2′ or 3′ succinate of a ribonucleoside attached to a solid support. The coupling of component 1 and 5′-OH-n-protected-2′,3′-protected-nucleoside are also achieved in solution phase in the presence of an activator to lead to dimers and oligoribonucleotides, followed by oxidation (3′→5′ direction synthesis), also lead to protected dinucleoside having a 3′-5′-internucleotide linkage, Ogilvie, K. K., Can. J. Chem., 58, 2686, 1980.
The N-acetyl guanine protecting group offers great potential in RNA synthesis of defined sequence based on our invention as outlined here.
This group can be utilized in conjunction with various 2′-protecting groups required for RNA synthesis. The most widely utilized 2′-protecting, tert-butyl-dimethylsilyl, which has been extensively developed by Ogilvie and coworkers as 2′-hydroxy protecting group for oligo ribonucleotide synthesis (Ogilvie, K. K., Sadana, K. L, Thompson, E. A., Quilliam, M. A., and Westmore, J. B Tetrahedron Letters, 15, 2861-2864, 1974; Ogilvie, K. K., Beaucage, S. L, Entwistle, D. W., Thompson, E. A., Quilliam, M. A., and Westmore, J. B. J. Carbohydrate Nucleosides Nucleotides, 3, 197-227, 1976; Ogilvie, K. K. Proceedings of the 5th International Round Table on Nucleosides, Nucleotides and Their Biological Applications, Rideout, J. L., Henry, D. W., and Beacham L. M., III, eds., Academic, London, pp. 209-256, 1983).
These studies subsequently led to continued developments of methods which were amenable to both solution and solid phase oligonucleotide synthesis, and the first chemical synthesis of RNA molecules of the size and character of tRNA (Usman, N., Ogilvie, K. K., Jiang, M.-Y., and Cedergren, R. J. J. Am. Chem. Soc. 109, 7845-7854, 1987; Ogilvie, K. K., Usman, N., Nicoghosian, K, and Cedergren, R. J. Proc. Natl. Acad. Sci. USA, 85, 5764-5768, 1988; Bratty, J., Wu, T., Nicoghosian, K., Ogilvie, K. K., Perrault, J.-P., Keith, G. and Cedergren, R., FEBS Lett. 269, 60-64, 1990). The literature has been amply reviewed in subsequent excellent publication: Gait, M. J., Pritchard, C. and Slim, G., Oligonucleotides and Their Analogs: A Practical Approach (Gait, M. J., ed.), Oxford University Press Oxford, England, pp 25-48, 1991. Other protecting groups which have been lately employed for RNA synthesis are; bis(2-acetoxyethyl-oxy)methyl (ACE), Scaringe, S. A., Wincott, F. E., Caruthers, M. H., J. Am. Chem. Soc., 120: 11820-11821, 1998; triisopropylsilyloxy methyl (TOM), Pitsch, S., Weiss, P. A., Jenny, L., Stutz, A., Wu, X., Helv. Chim. Acta. 84, 3773-3795, 2001 and t-butyldithiomethyl (DTM) (structure 16), Semenyuk, A., Foldesi, A., Johansson, T., Estmer-Nilsson, C., Blomgren, P., Brannvall, M., Kirsebom, L. A., Kwiatkowski, M., J. Am. Chem. Soc., 128: 12356-12357, 2006 have been introduced.
Recently the scientists at ChemGenes have developed the method of RNA synthesis in reverse direction (5′→3′ direction), for efficient incorporation of many ligands and chromophores conveniently and efficiently at the 3′-end of RNA molecules and a recent publication, Srivastava, S. C., Pandey, D. P., Srivastava, N., Bajpai, S. P., Nucleic Acids Symposium Series No. 52, 103-104, 2008 (structure 20, 21 & 22). Appropriately N-acetyl protected guanine can nucleosides, deoxy and ribo are envisaged to be synthesized and DNA and RNA synthesis is proposed to utilize the benefits which reverse DNA and RNA synthesis offer. (US patent application entitled “RNA Synthesis—Phosphoramidites for Synthetic RNA in the Reverse Direction, and Application in Convenient Introduction of Ligands, Chromophores and Modifications of Synthetic RNA at the 3′-End” by Suresh C. Srivastava et al. filed on Sep. 8, 2009, and US patent application entitled “Synthesis of N-Fmoc Protected Deoxy Nucleosides, Ribo Nucleosides . . . ” by Srivastava et al. filed on Nov. 30, 2009)
A novel 2′-protecting group, acetal levulinyl ester (ALE) (structure 15 has been recently proposed (J. G. Lackey and M. J. Damha, Nucleic Acids Symposium Series, No. 52, 35-36, 2008). Similar to this protecting group another 2′-labile protecvting group based on similar chemical nature, 2′-O-acetal ester, pivaloyloxy methyl which has been found mild 2′-O protercting group, T. Lavergne, A. Martin, F. Debart, J-J Vasseur, Nucleic Acids Symposium Series No. 52-51-52, 2008. The base protecting group used by these authors was n-acetyl and tbPAC. This gives additional credence to our process. And n-acetyl for RNA synthesis with other 2′-protecting groups or 2′-modification would be an ideal group for deprotection under mild basic condition in shorter period of time.

Chemically modified RNA have been synthesized having modified arabino sugars, 2′-deoxy-2′-fluoro-beta-D_arabinonucleic acid (FANA; structure 17)) and 2′-deoxy-4′-thio-2′-fluoro-beta-D_arabinonucleic acid (4′-Thio-FANA; structure 18) into sequences for SiRNA activities, Dowler, T., Bergeron, D., Tedeschi, Anna-Lisa, Paquet, L., Ferrari, N., Damha, M. J., Nucl. Acids Res., 34, 1669-1675, 2006. Amongst several new 2′-protecting group chemistry which have been developed, the 2′-protecting 2-cyanoethoxymethyl (CEM) (structure 19) has been shown for producing very long RNA also carries out RNA synthesis in conventional (3′→5′ direction). However the quality of these long RNA remain in question.
